Encapsulation of microparticles in teardrop shaped polymer capsules of cellular size

ABSTRACT

Microparticles such as propagules of eukaryotic biocontrol agents are encapsulated in cellular-scale polymer capsules that have a diameter similar to normal eukaryotic cells in a range of about 10 μm to about 400 μm. The microparticles are encapsulated by adding a hydrophobic dispersion medium such as a mixture of chloroform and hexane or a mixture of corn oil and n-hexadecane having a specific gravity of about 1 and containing an emulsifier such as lecithin to an aqueous suspension of the microparticles and a polymer matrix precursor such as alginate, agitating vigorously to form a stable emulsion of microscopic globules containing a microparticle, and adding the emulsion to an aqueous solution containing a polymerizing agent such as calcium chloride to polymerize and precipitate the globules to form microparticles encapsulated in polymer matrix capsules that may be of a teardrop shape having a length of 40-200% longer than the diameter. Precipitation of the globules is regulated by substantially matching the Specific Gravity of the hydrophobic dispersion medium and the aqueous suspension. The microparticle may be a viable propagule of a weed pathogenic fungus to provide a herbicidal composition.

FIELD OF THE INVENTION

This invention relates to a method for the cellular-scale encapsulationof microparticles, and to microparticles encapsulated according to themethod.

BACKGROUND OF THE INVENTION

Recently, there has been considerable interest inencapsulation/immobilization of microparticles, including living cells,propagules of living cells, bacteria, viruses, fungi and the like.

Moreover, it is often desirable to formulate biological control agentsso that their propagules are encapsulated and provided with water andnutrients necessary for growth. In this context, the necessity toovercome dew period requirements of weed pathogens is critical.Encapsulation can protect propagules from loss of viability due todesiccation, damage by UV light, and other environmental stresses. Itcan also aid in packaging inoculum in a form that is easier tomanipulate and harder for pests to detect.

Simple and sometimes effective anti-desiccant formulations have spannedthe range from oils to polysaccharide gets and guns, etc. There are araft of papers on the subject, please see Auld & Morin, 1995¹ and Greenet al. (1998)² for comprehensive overviews. There are various polymerencapsulation methods that have been derived for bacteria and othersmall targets. In biological control research, most attempts toencapsulate spores and other cells have involved their inclusion invarious kinds of macroscopic polymer granules (alginates, pastas), e.g.see Connick et al., 1991³. In biological control of weeds, invertemulsions are used with increasing frequency, because they can overcomethe requirement for a dew period, e.g. see Connick et al., 1991⁴.

The above methods all have limitations. Simple antidessicant gels, etc.have had limited success owing to the high viscosity needed to achieveadequate water-holding properties. In other words, such formulationscannot be easily sprayed, and require far more material than iseconomically feasible to apply. Invert emulsions suffer similarproblems, in that they are bulky, and costly in terms of the amount ofmaterial that must be applied. They can cause collateral damage becausethey can be phytotoxic in their own right, and they require specialequipment for application due to their high viscosity. In addition,invert emulsions must be prepared shortly before application, and do notallow for freeze-drying or other types of long term storage. Regardingdry formulations, macroscopic granules containing eukaryotic cellscannot be reduced to a microscopic size without crushing and killingcells. This is unfortunate, because macroscopic granules cannot beeasily sprayed and they do not efficiently distribute inoculum or othermaterials because of their relatively low surface area to volume ratio.Vapor coating methods are also impractical in our experience, due to thecost of the method, the cumbersome equipment required, the exposure ofcells to relatively high heat during some coating methods, the dry,piecemeal nature of coatings produced at lower heats, and the largersize of the particles produced. A method is needed to make formulationsa more intrinsic part of cells, hence encapsulation.

Currently applicable encapsulation or immobilization techniques tend toproduce polyacrylamide or alginate beads in the range of 0.5-1 mm indiameter, too large to be of practical use for cellular-scaleapplications. They tend to be expensive or unwieldy and rely on methods,equipment, or chemicals that are fairly specialized. It is possible toplace aqueous droplets containing acrylamides or other monomers intodispersion media composed of hydrophobic solvents or oils, wherein thedrops are held in place as spheres while the monomers polymerize. Thismethod comes closest to our approach, e.g. see Nilsson et al., 1983⁵,also see U.S. Pat. No. 4,647,536, but the technique is unwieldy, mostlybecause the polymerization is proceeding as the drops are formingglobules in the solvent.

A major advantage of the present methodology is that we avoid thecomplicated prior art procedures to extract the capsules from dispersionmedia, i.e. the capsules form on their way out of the dispersion medium,killing two birds with one stone. This is not only a unique method, butalso results in a unique characteristic—the capsules are not encumberedby any significant (visible in the compound microscope) oil coating.There are occasionally some small oil droplets that can be seen in andoutside the capsules, but these are a very small portion of the overallmaterial that is produced. Freeing the capsules from the dispersionmedium in this manner is desirable for a number of reasons. First,materials with an oily consistency may be difficult to manipulate,concentrate, or formulate for practical applications. Second, thepresence of significant amounts of the dispersion medium may affect theperformance or behavior of the encapsulated material. Third, thepresence of an extra component could complicate legal registration orother types of regulatory approval necessary for commercialization ofproducts. Fourth, on larger scales, aqueous solutions are cheaper thanoil solutions. There is another advantage to polymerizing upon exit fromthe dispersion medium: while Nilsson's stirring method avoids adhesionor bonding (perhaps even fusion) of capsules polymerizing inside thedispersion medium, our method does not require such fine control overagitation, and is thus more reliable and easier to reproduce,particularly if the method is to be scaled-up in volume.

SUMMARY OF THE INVENTION

It is an object of the present invention to encapsulate microparticlese.g. propagules of eukaryotic biocontrol agents with cellular-scalepolymer capsules, via a novel, inexpensive method that encapsulates withas much as 100% efficiency.

According to one aspect of the invention, a method is provided forcellular-scale encapsulation of microparticles, comprising:

(i) providing an aqueous suspension of the microparticles and a polymerprecursor, the polymer being biodegradable and not requiring heat orpressure to form,

(ii) adding a non-cytotoxic, hydrophobic dispersion medium containing anemulsifier, to form an aqueous dispersion,

(iii) agitating vigorously to form a stable emulsion of microscopicglobules including a microparticle and the polymer precursor, and

(iv) adding the suspension to an aqueous solution containing apolymerization agent/catalyst, to polymerize and precipitate theglobules and form polymer encapsulated microparticles of cellular scaledimensions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photomicrograph of a capsule containing macroconidia ofFusarium avenaceum (Fr.) Sacc., generated via the corn oil method. Theblack scale bar is 100 μm long.

FIG. 2 is a photomicrograph of a similar capsule, with germinatingconidia. The black scale bar is 100 μm long.

FIG. 3 is a graph illustrating the effect of dispersion mediumcomposition on capsule diameter. The regression line shows a significant(P<0.0001) trend, and the dashed lines show 95% confidence limits forthe trend.

FIG. 4 is a graph illustrating the relationship between capsule diameterand capsule length. The regression line shows a significant (P<0.0001)trend.

FIG. 5 is a graph illustrating the effects of dispersion mediumcomposition (ratio of corn oil:n-hexadecane) and alginate concentrationon the diameter of capsules produced by the corn oil method.

FIG. 6 is a graph illustrating the effects of dispersion mediumcomposition (ratio of corn oil:n-hexadecane) and alginate concentrationon the diameter and shape of capsules produced by the corn oil method.The ratio of capsule length:width is a measure of capsule elongation,with a value of 1 corresponding to a spherical shape, and higher valuescorresponding to elongation. Values of zero indicate that precipitationdid not occur in the particular treatment combination.

FIG. 7 is a graph illustrating the effects of emulsifier (lecithin)concentration on the diameter of capsules produced by the corn oilmethod.

FIGS. 8 and 9 are graphs illustrating the effects of relative humidityand CaCl₂ concentration on the germination of plain (control), orencapsulated conidia of Fusarium avenaceum, respectively.

FIG. 10 is a photomicrograph of a capsule generated by the corn oilmethod, with conidia of Fusarium avenaceum germinating in 20% relativehumidity, illustrating that the germ tubes remain inside the capsuleuntil conditions are more favorable. The black scale bar is 100 μm long.

FIG. 11 is a photomicrograph of the hyphae of Chondrostereum purpureum(Pers. Ex Fr.) Pouzar encapsulated using the corn oil method,germinating on a microscope slide exposed to the air. The black scalebar is 100 μm long.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The method involves the use of stabilized (with a suitable emulsifier)emulsions in conjunction with an aqueous solution containing apolymerizing agent. To form the emulsion, the cells or microparticles tobe encapsulated are suspended in aqueous solutions containing polymerprecursors, and this is added to a hydrophobic dispersion medium(solvent) containing the emulsifier e.g. lecithin. After vigorousagitation, a stable emulsion of microscopic globules containing thepolymer precursor and the material to be encapsulated form within thehydrophobic dispersion medium. To polymerize these globules, theemulsion is layered onto the solution containing the aqueouspolymerizing agent and the globules are allowed to precipitate into thissolution and polymerize. This method is generally applicable to a widerange of chemical solvents (see below), and is not restricted to thesolvents tested in the experiments outlined below. Because it ispossible to include air bubbles in emulsions, the dispersion medium neednot necessarily be lighter than water, and could, in fact, be locatedunder the catalyst (polymerizing agent) solution as globules rise andpolymerize to form buoyant capsules. The method is also applicable to avariety of microparticles and cellular material, including yeast cells,somatic cells and sexual cells. Of particular interest is theencapsulation of viable fungal propagules, useful as biocontrol agents,and synthetic spores.

The key factors are 1) The presence of a stabilized emulsion containingaqueous globules of cellular-scale dimensions; 2) The presence ofnon-miscible dispersion medium and water phases with low surface tensionat their interface; 3) The use of fast-polymerizing materials toencapsulate, and 4) bead (globule) formation before polymerization. Forencapsulation of living materials, the use of certain solvents (e.g.n-hexadecane) to adjust globule size and interface surface tension ispreferred to preserve cell viability.

Cellular-scale is defined here as a size range similar to the diameterof normal eukaryotic cells, i.e. ranging from approximately 10 μM toapproximately 400 μM.

Summary of Alternative Compounds Useful in Encapsulation Technique

A. Encapsulation polymers

TABLE 1 A list of polymers useful in encapsulation technique. PolymerOrganism Reference pregelatinized cornstarch Bacillus McQuire and Shasha(1990) J. Econ. thuringiensis Entomol. 83:5, 1813-7 Alginate BacillusVijayan and Balaraman (1995) Southeast thuringiensis Asian J. Trop. Med.Public Health 26:1, 183-7 Alginate Bacillus Elcin (1995) J.Microencapsul. 12:5, 515-23 thuringiensis polylactic-coglycolic acidProtein Yang and Cleland (1997)J. Pharm. Sci. 86:8, 908-14alginate-polylysine None De Vos, De Haan, Schilfgaarde (1997)Biomaterials 18:3, 273-8 Desaminotyrosinetyrosylhexyl ester with NoneErtel Kohn, Zimmerman, Parsons (1995) J. phosgene Biomed Mater Res.29:11, 1337-48 poly(lactic acid-co-glycolic acid) None MeneiBoisdron-Celle, Croue, Guy, Benoit (1996) Neurosurgery, 39:1, 17-23derivatized alpha-amino acids Ovalbumin Vaccine (1996) 14:8, 785-91polyacrylonitrile-sodium methallylsulphonate Hepatocytes Biomaterials,1995, 16:10, 753-9 cellulose sulfate, poly(methylene-co- Living cellsNat. Biothechnol. 1997 15:4, 358-62 guanidine)hydrochlorideagarose/poly-(styrene sulfonic acid) PC12 cells Exp. Neurol. 1996 138:1,169-75 (agarose/PSSa) Methacrylic acid and dimethylaminoethyl Raji CellsBiomaterials, 1995 16:4, 325-35 methacrylate water-insolublehydroxyethyl methacrylate- PC12 cells Biomaterials, 1996, 17:3, 267-75methyl methacrylate copolymer and polyethylene glycolpoly(2-hydroxyethyl methacrylate) and poly(2- Insulin releasing J.Biomater. Sci. Polym. Ed. 1997, 8:8, 575- hydroxyethylmethacrylate-co-methyl cells 86 methacrylate) alginate emulsified invegetable oil Bacillus J. Microencapsul. 1997, 14:5, 627-38Calmette-Guerin hydroxyethyl methacrylate-methyl methacrylateErythrocytes Journal of Biomedical Materials Research copolymer(HEMA-MMA) 24(9), 1241-1262 sodium alginate, kaolin clay CercosporaPhytopathology 75(2), 183-185 kikuchii Calcium alginate Live cells andJournal of Microencapsulation Vol. 12, no. 3, enzymes 255-262Alginate/poly-L-lysine (alg/PLL), nylon or Lactoccus lactis J.Microencapsulation Vol. 11, no. 2, 189- crosslinked polyethyleneimine(PEI) subsp. cremoris 195 membranes Biodegradable mixtures of pectin andPolyvinyl None http://www.arserrc.gov/TechPectinPVA.htm alcohol (U.S.Pat. app. 08/529,299 Alginate-polylysine Isolated rat Biomat., Art.Cells & Immob. Biotech. 19(4), hepatocytes 675-686 Agar

Some additional polymers could be incorporated in Table 1. They can beformed and molded without elevated temperature or pressure and couldtherefore be used to encapsulate cells. Even if they do not polymerizeas quickly as alginate, the alginate capsules could be used to holdthese other polymers in place until they harden. The general classes ofpolymers not requiring heat or pressure to form include:

Epoxy plastics

Polyesters

Polyurethanes

Silicone plastics

Acrylamids or polyacrylamides

Note that any of the preceding polymers (Table 1 and list) could becombined in mixtures. Note also that the polymer may degrade or solidifywith time, or with exposure to light, heat, chemical signals, orenvironmental signals, thereby affecting the biological or physicalperformance of the encapsulated material. Other biological polymers havebeen frequently listed in the literature as adjuvants for biologicalcontrol agents. Examples of general classes of biological compounds thatcould be useful alone or in mixtures to create biodegradable polymercapsules for these agents or other materials could include:

Carageenans

Fibrins

Sugars and polysaccharides, including starches

Amino acids and proteins

Glycoproteins and peptidoglycans

Nucleic acids, DNA, and RNA

Fatty acids and lipids

Lipoproteins and liposaccharides

Organic polymers containing mixtures of any of the above compounds

B. Emulsifiers

Common water-in-oil emulsifiers that could be used for theemulsification technique include:

General groups:

Lecithins

Glycerides (including mono- and di-glycerides)

Polysorbates

Glyceryl esters

Sorbitan esters

Cholesterol and other steroids

Acacia extracts

Gelatins

Phospholipids (bilayer forming)

Specific examples of emulsifying agents from “Remington's PharmaceuticalSciences” (1990) include:

Sorbitan trioleate

Sorbitan tristearate

Propylene glycol monostearate

Sorbitan sesquioleate

Glycerol monostearate (non self-emulsifying)

Sorbitan monooleate

Propylene glycol monolaurate

Sorbitan monostearate

Glyceryl monostearate (self-emulsifying)

Sorbitan monopalmitate

Sorbitan monolaurate

Polyoxyethylene-4-lauryl ether

Polyethylene glycol 400 monostearate

Polyoxyethylene-4-sorbitan monolaurate

Polyoxyethylene-20-sorbitan monooleate

Polyoxyethylene-20-sorbitan monopailitate

Polyoxyethylene-20-sorbitan monolaurate

Polyoxyethylene-40-stearate

Sodium oleate

Sodium lauryl sulfate

Lecithin, bilayer forming lipids and cholesterol are preferred. Lecithinis most preferred.

C. Alternative oils and solvents (dispersion medium)

Almost any naturally occurring biologically derived oil can be used forthe oil portion of the dispersion medium, as most will be bothhydrophobic and non-toxic to cells. Synthetic oils or petroleum basedoils could be useful in encapsulation of non living targets, but most ifnot all would probably have some toxicity to living cells. It is to beexpected that non-toxic synthetic oils that mimic naturally occurringbiologically derived oils can be used, but they would probably be moreexpensive.

Regarding solvents, it would be difficult to find many ordinary typesthat would not break down cell membranes and kill cells. However,because methylation and other ways of creating chemical derivativesprovides an opportunity to design more solvents than we can list in onechart, we will simply describe the characteristics the solvent shouldhave to be useful in the encapsulation approach:

Hydrophobic (hydrocarbons), miscible in oils

Structure (chains, branches, or cyclic rings) arranged so that thespecific gravity is relatively close to the specific gravity of water

No reactive groups in the molecule that would be toxic to cells (nochlorine, carboxyl, hydroxyl, etc. groups).

Molecules large enough that they don't easily penetrate biologicalmembranes

Relatively non-viscous

METHOD

Note that fungal propagules used throughout these methods were producedusing standard microbiological culturing methods, e.g. consult Winder,1996⁶, the disclosure of which is incorporated herein by reference. Alsonote that the percentage values for all solution concentrations werecalculated on a volume:volume basis.

Case 1: Alginate capsules using a Chloroform:Hexane solvent system.

One of the main considerations in the formation of an emulsion is thetendency of the droplets to cream or precipitate. The ideal emulsion isone in which the individual droplets remain individual and neither sinknor float. One means of achieving this is to form the emulsion dropletsin a medium with substantially the same specific gravity as the dropletsthemselves. Rapid mixing, homogenization, sonication, etc. can emulsifywater and organic solvent, but the droplets usually rapidly float to thesurface (cream) and fuse until eventually one is left with a two-phasesystem. Emulsifiers such as lecithin slow this process down somewhat. Itwas found that if one mixed chloroform with the lighter hexane, itresulted in a solvent system which was the same specific gravity as a0.5% aqueous alginate solution emulsified into it. This required amixture ratio of 1.5 mL hexane to 0.5 mL chloroform. Vortexing severaldrops of 1% sodium alginate solution added to 10 mnL of the solvent leadto rapid breakdown of the water phase and separation. When lecithin wasincluded (25 mg to start with, but this can be reduced), it was possibleto form small droplets which were very stable. By adjusting the ratio ofchloroform to hexane it was possible to completely suspend thesedroplets, or cause them to sink or cause them to float. In all cases,the droplets did not fuse.

To polymerize the droplets, the organic solvent containing the alginatedroplets was layered over a 1 mL solution of 0.1 M calcium chloride(CaCl₂) in a test tube (calcium ions cause liquid alginate toimmediately polymerize and harden). During the first trial, the CaCl₂contained about 1 μg of A23187 (a calcium ionophore) to enhancepenetration into the capsules, but the ionophore was found to beunnecessary in subsequent attempts. The emulsion droplets slowlyprecipitated from the solvent phase and polymerized upon crossing thesolvent/CaCl₂ solution interface. These droplets collected on the bottomof the test tube and were recovered with a Pasteur pipette and washedwith water to remove any residual solvent.

The resulting capsules were all less than 500 μm in diameter. As seen inFIG. 1, most were uniformly teardrop-shaped, with symmetrical morphologyand tails, since the polymerization proceeded quickly. Conidia (spores)of the fungus Fusarium poae (Peck) Wollenw. (a potential fungalbiocontrol agent for marsh reed grass) were encapsulated into thealginate beads with 100% efficiency. In subsequent trials, it was foundthat the concentration of alginate (viscosity of the emulsifieddroplets) affected the size of the beads formed. Lower alginateconcentrations caused smaller beads (100 μm or less) to form. Loweralginate concentrations required higher hexane:chloroform ratios toachieve precipitation of the droplets into the CaCl₂ phase. The amountof lecithin used in the first experiment (25 mg) was far in excess ofwhat was necessary to achieve a stable emulsion. In subsequent trials,0.1 mg or less was needed.

At this point, the feasibility of encapsulation using the method wasdemonstrated. However, the chloroform was probably toxic to the spores,as they did not germinate. For applications involving living cells, themethod required adaptation. It was possible to address this, since thefeature critical to achieving adequate precipitation of capsules is theregulation of emulsification and interface with the aqueous polymerizingsolution, and not the particular solvents used.

Case 2: Corn oil: hexadecane solvent system.

It was found that a stable emulsion could be formed by emulsifyingalginate solutions in a dispersion medium of a mixture of cornoil:n-hexadecane containing lecithin. The resulting droplets alsopolymerized by precipitation into a CaCl₂ 0.1 M solution as mentionedabove. Hexadecane was chosen because it is the solvent used by Crooks etal. (1990)⁷ for the formation of MAA copolymer beads encapsulatingcells, where it is harmless to cell viability. In this case, theemulsion was stabilized by the lecithin, while the size and buoyancy ofcapsules was controlled by varying the corn oil:n-hexadecane ratio.Higher proportions of corn oil produced small, buoyant capsules whichdid not precipitate into the aqueous phase, while higher proportions ofhexadecane resulted in unstable emulsions which produced capsules oflarger than cellular proportions (Table 2). The best results wereobtained with a ratio of about 60% corn oil, 40% hexadecane. Theemulsions for the dilution series experiment were formed using 10 mLsolvent, 9 large drops of conidial solution, and 1 small drop oflecithin.

TABLE 2 Formation of capsules in various solvent ratios. In all ratioswhere capsules formed, conidial germination was ca. 100%. Ratios in boldfont are desirable for encapsulation. Corn oil: n-hexadecane ratioCapsule characteristics 0:1 Most >500 μm 1:9 Most >500 μm 1:4 Most >500μm 3:7 Most >500 μm 2:3 Most <300 μm 1:1 Most <300 μm 3:2 Most <100 μm7:3 Most <200 μm 4:1 No yield 9:1 No yield 1:0 No yield

The capsules formed by this method can be very small (<100 μm diameter)(FIG. 1). Macroconidia (spores) of Fusarium avenaceum (another potentialbiocontrol agent of marsh reed grass, q.v. Winder 1997⁶) encapsulated bythis method in alginate beads were viable and germinated at normalpercentages, with germ tubes rupturing out of the capsules—exposure tohexadecane does not appear to inhibit spore germination (FIG. 2)

A further study (test tube assay) was done to more precisely determinethe capsule size and encapsulation efficiency, as a result of variousratios of corn oil and n-hexadecane.

Specifically, dispersion media consisting of 0, 10, 20, 30, 40, 50, 60,70, 80, 90 and 100% corn oil versus n-hexadecane were prepared (eachcontaining 0.1% lecithin). 10 mL of each medium were poured into a testtube (3 replications per medium type). The media were vortexed for 5seconds with 10 drops of an aqueous 1% sodium alginate solutioncontaining 1 mg/100 mL Evan's Blue dye for visualizing theprecipitation. The resulting emulsions were layered over 5 mL of 0.01 MCaCl₂ solution and the diameter of the first 50 capsules harvested fromeach tube was measured with a microscope. Oil concentration of less than50% tended to favor production of larger droplets in the emulsion, andfusion of the capsules into irregular masses as they precipitated.Higher oil concentrations favored smaller capsules (see FIG. 3). Oilconcentrations greater than 70% did not allow for precipitation, and thealginate droplets were very small (<50 microns) and well mixed with thedispersion medium under those conditions. The addition of liquid soap toan 80% system induced a few capsules to precipitate, indicating thatsurface tension at the catalyst (polymerizing agent)/dispersion mediuminterface is partially responsible for regulating the occurrence ofprecipitation (the other factor is probably droplet size). This alsomeans that any surface tension reducing surfactant could be added to thecatalyst solution as part of any final method, as a way to reducen-hexadecane consumption. Precipitation in the 70% systems was generallyless rapid, and one 70% system did not form precipitate, indicating that70% corn oil:n-hexadecane is very near the threshold beyond whichprecipitation will not occur, i.e. in the absence of any surfactants inthe catalyst solution. Overall, we attribute the effect of oilconcentration on droplet size to the combined effects of viscosity andspecific gravity.

A further factorial study (test tube assay) was done to more preciselydetermine capsule diameter and length in relation to the proportion ofcorn oil:n-hexadecane and the percentage of alginate in theprecipitating droplets. Specifically, dispersion media consisting of 10,30, 50, and 70% corn oil versus n-hexadecane were prepared (eachcontaining 0.1% lecithin). Each medium was poured into separate testtubes (5 mL per tube). Aqueous solutions of 0.5%, 1%, 1.5%, and 2%aqueous sodium alginate were prepared, and for each solution, four dropswere placed into a separate tube of each medium type, resulting in 16combinations of medium type and alginate concentration. The media werevortexed for 5 seconds and the resulting emulsions were layered over 5mL of 0.01 M CaCl₂ solution. The diameter and length of the first 14capsules harvested from each tube was measured with a microscope. Ingeneral, capsule length tended to be about 40% longer than capsulediameter, with most capsules being less than 200% longer, and nocapsules being more than 700% longer FIG. 4). Oil and alginateconcentrations affected the size of emulsified globules (FIG. 5), withno precipitation occurring for high (70%) proportions of oil combinedwith high (2%) or low (0.5%) proportions of alginate. We attribute theseresults to the lower specific gravity of 0.5% alginate at one extreme,and the higher viscosity of 2% alginate at the other. In low (10%)proportions of oil combined with 1% alginate, the ratio of capsulelength to width was significantly greater (FIG. 6). We attribute theelongation of these capsules to reduced surface tension at the interfacebetween the dispersion medium and the aqueous catalyst solution,combined with a slightly larger capsule size and reduced capsulebuoyancy. As surface tension is reduced, the larger globules wereprecipitating further into the aqueous catalyst solution before remnantsof the dispersion medium retracted to allow polymerization. Thisresulted in elongation of the teardrop shape.

A further study (test tube assay) was conducted to determine the effectof emulsifier (lecithin concentration) on capsule size and theefficiency of capsule production. 10 mL of a dispersion mediumconsisting of 1:1 corn oil:n-hexadecane was dispensed into test tubes,and lecithin was added to make dispersion media containing either 0,0.17, 0.33, or 0.50% lecithin. For each type of dispersion medium, therewere three replications. 9 drops of an aqueous solution of 1% sodiumalginate were added to each tube and vortexed. Capsules were generatedby layering the medium over an aqueous solution of 0.01M CaCl₂, andcapsule diameters and lengths were measured as previously described.Increasing proportions of lecithin controlled capsule size bystabilizing the emulsion, thus preventing fusion of globules andallowing smaller capsules to precipitate (FIG. 7). Although a fewcapsules formed in the absence of lecithin (FIG. 7), production wasalmost nil, since most of the alginate fused at the interface betweenthe dispersion medium and the aqueous catalyst solution. It is thereforenecessary for an emulsifier to be present if substantial numbers ofindividual capsules are to be formed. With a small (0.17%) amount oflecithin, a moderate amount of capsules were produced. With greaterproportions of lecithin, the efficiency of production increased toinclude nearly all of the alginate. It is evident that proportions oflecithin greater than 0.17% are desirable for obtaining maximumstability of the emulsion and therefore efficiency of production (FIG.7). The proportion of lecithin had no significant effect on the capsulelength:width ratio.

We expect that the actual upper limit is the amount that would begin tosignificantly increase viscosity or alter the specific gravity orsurface tension of the dispersion medium. The precise achievable limitwould be relative to other factors. Certainly, 100% lecithin would bemore of a paste than a liquid, and precipitation would not occur. Theactual upper limit would depend greatly on the proportion of oil in thedispersion medium and the quality (purity, type) of lecithin.

A further study (test tube assay) was performed to determine the effectof catalyst (CaCl₂) concentration on capsule shape and productionefficiency. A dispersion medium consisting of 1:1 corn oil:n-hexadecanewas prepared, containing 0.2% lecithin. 5 mL of the dispersion mediumwas dispensed into six separate test tubes, and 4 drops of 1% aqueoussolution of sodium alginate were added to each tube. Tubes were vortexedas previously described and for each tube the dispersion medium waslayered over either a 0.01 M or 1.0 M aqueous solution of CaCl₂ (threetubes per CaCl₂ concentration). Capsule lengths and widths were measuredas previously described. The higher concentration of catalyst nearlyhalved capsule diameter, from 158±19 μm in 1.0 M CaCl₂ to 85±6 μm in0.01 M CaCl₂. Efficiency was also nearly 100% and capsules were muchless irregular in shape at the higher concentration of CaCl₂. Weattribute these effects to changes in surface tension and rate ofcatalyst diffusion into the capsules. At higher concentrations of CaCl₂,surface tension of the aqueous catalyst solution is reduced, so thatglobules more readily precipitate across the interface with thedispersion medium and polymerize rapidly before they fuse with nearbyglobules. There was no significant effect of catalyst concentration onthe length:width ratio of the capsules.

We expect that the upper limit is the amount that would begin to eithersignificantly increase viscosity or alter the specific gravity of thecatalyst solution, or the amount which would begin to significantlyreduce the viability or activity of encapsulated cells. Again, theprecise achievable limit will also depend on the type of dispersionmedium, and the type of cells that are encapsulated.

It will be appreciated that the encapsulated materials may includecrystals, liquid droplets, gas bubbles, other capsules, or othermicroparticles with chemical, electrical, or physical activity.

Moreover a wide variety of substances can be included with theencapsulted material. In many types of formulations it is usual toinclude substances which may affect the biological or physicalperformance of the encapsulated material (see the Green et al., 1998².Some general classes of substances that could be used to affect thebehavior, appearance, or taste of encapsulated biomaterials arenutrients, preservatives, buffers, growth factors, toxins, enzymes,flavors, odors, pigments, dyes, chemical signals, adhesives, and gases.In biological control, it may be very advantageous to encapsulate theabove kinds of substances along with biomaterials, because the materialswould remain concentrated in the capsule even after dissemination intothe environment.

Protective qualities

As research on this encapsulation method progresses, amendment to thecorn oil method outlined above may become necessary as we discover waysto improve the capsule design and resistance of inoculum to desiccation.However, we already see evidence that the capsules will provide someprotection from desiccation, in conjunction with the presence of CaCl₂in the inoculum solution. We have assayed the germination ofencapsulated macroconidia of F. avenaceum at 20° C. in drops placed arange of small glass humidity chambers. The relative humidity (RH) inthe small glass chambers was controlled according to the method ofWinston and Bates (1960)⁸, the disclosure of which is incorporatedherein by reference, with saturated chemical solutions, which produce apartial pressure of water vapor in a sealed space which is readilycalculable. There were four replications (separate drops) per humidityvalue per trial—statistically significant (P<0.001) effects aresummarized as follows. Conidia were encapsulated as in the corn oilmethod above. Encapsulated conidia in 0.1 M CaCl₂ germinated readily athigher RH values, but lost viability at 85% RH. We attribute this togreatly increased concentrations of CaCl₂ at lower RH values as thedrops evaporated. F. avenaceum conidia did not germinate in pure water.We attribute the better germination in capsules to the presence ofnutrients in the alginate, and the osmotic effect of the CaCl₂ (FIGS. 8and 9). When we encapsulated the conidia using 0.01 M CaCl₂, a differentpicture emerged. The hygroscopic calcium salt allowed for a scantgermination in controls at higher RH values, and maintained enough waterfilm for a very few conidia to germinate even in the driest conditions.This was not unexpected, since evaporation was not instantaneous andvarious salts are known to stimulate germination of spores in manyfungal species. This does not, therefore, limit the scope of theinvention. Germination was much greater for encapsulated conidia, whereconidia germinated in substantial numbers even under fairly dryconditions. A peak occurred at 55% RH, which we attribute to differencesin the hygroscopic properties of the partially evaporated droplets.Below 55% RH, germ tubes remained inside the capsules (FIG. 10). Theseresults demonstrated the potential for practical uses of the method.

Case 3

The corn oil method stated in case 2 above was used to engineerartificial microscopic spores of the fingus Chondrostereum purpureum.The dispersion medium consisted of a 6:4 ratio of corn oil:n-hexadecane,with 0.2% lecithin as an emulsifier. Hyphal clumps of Chondrostereumpurpureum were produced in a liquid malt extract culture using standardmicrobiological methods. 10 mL of the culture was mixed with 100 mL of1.0% sodium alginate, and 9 drops of the resulting suspension were addedto 10 mL of the dispersion medium in a test tube. The tube was vortexedfor 20 seconds, and the medium was layered over 10 mL of 1.0 M CaCl₂.Drops containing the resulting capsules were placed on microscope slidesexposed to the air and examined for viability after 24 h. FIG. 11 showshyphae of Chondrostereum purpureum germinating from one such capsule(all hyphae were internal to capsules prior to incubation). This resultis significant in two respects. First, the hyphae of C. purpureum inliquid culture are thin-walled and fragile. In effect, our novelencapsulation technique has made a small clump of fragile hyphae into arobust spore. This may very well be the first instance of a deliberatelyengineered microscopic fungal spore. The other significance is that C.purpureum is being commercially developed as a biocontrol agent (see ourcommonly assigned U.S. Pat. No. 5,587,158). Moreover, it is difficult tospray existing hyphal formulations. Keeping in mind that spores of C.purpureum and many other types of fungi are difficult to mass-produce,we believe that this result demonstrates that we can make artificialspores for virtually any fungus, as long as we can grow the hyphae.Fungi that grow in artificial culture but do not produce usefulquantities of durable spores include sterile deuteromycetes, biotrophicfungi, mychorrhizal mushrooms, and fungi which produce spores that arethin-walled and ephemeral. Using the above method, artificial sporescould be produced for all of these types of fungi.

REFERENCES CITED

1. Auld, B. and Morin, L. 1995. Constraints in the development ofbioherbicides. Weed Technology 9:638-652.

2. Green, S., Wade-Stewart, S., Boland, G., Teshler, M., and Liu, S.1998. Formulating microorganisms for biological control of weeds. Pages249-281 in Boland, G. and Kuykendall, L., Eds., Plant-microbeinteractions and biological control. Marcel Dekker, Inc., N.Y.

3. Connick, W., Boyette, D., and McAlpine, J. 1991. Formulation ofmycoherbicides using a pasta-like process. Biological Control 1:281-287.

4. Connick, W., Daigle, D., Quimby, P. 1991. An improved invert emulsionwith high water retention for mycoherbicide delivery.

5. Nilsson K., Birnbaum S., Flygare S., Linse L., Schroder U., JeppssonU., Larsson P.-O., Mosbach K. and Brodelius P. 1983 A general method forthe immobilisation of cells with preserved viability. Eur. J. Appl.Microbiol. Biotechnol., 17:319-326.

6. Winder, R. 1997. The in vitro effect of allelopathy and various fungion marsh reed grass (Calamagrostis canadensis). Canadian J. Botany75:236-241.

7. Crooks, C A. Douglas, J A. Broughton, R. L., Sefton M V. (1990)Microencapsulation of mammallian cells in a hema MMA copolymer effectson capsule morphology and permeability. Journal of Biomedical MaterialsResearch: 24(9):1241-1262

8. Winston P. and Bates, D. 1960. Saturated solutions for the control ofhumidity in biological research. Ecology 41: 232-237.

What is claimed is:
 1. A method for encapsulation of microparticles inpolymer matrix capsules having a diameter of about 10 μm to about 400μm, comprising: (i) providing an aqueous suspension of themicroparticles and a polymer matrix precursor, (ii) adding anon-cytotoxic, hydrophobic dispersion medium containing an emulsifier,to form an aqueous dispersion, (iii) agitating vigorously to form astable emulsion of individual microscopic globules containing amicroparticle and the polymer matrix precursor suspended therein, and(iv) adding the stable emulsion to an aqueous solution containing apolymerizing agent, to polymerize and precipitate the globules, to formmicroparticles encapsulated in polymer matrix capsules having saiddiameter and being of a teardrop shape baying a length of 40-200% longerthan its diameter, wherein precipitation of the globules is regulated bysubstantially matching the Specific Gravity of the hydrophobicdispersion medium and the aqueous suspension.
 2. A method according toclaim 1, wherein the dispersion medium contains an oil and an organicsolvent that regulates precipitation of the globules by varying theviscosity and specific gravity of the dispersion medium, andconsequently varying globule size.
 3. A method according to claim 1,wherein the emulsifier is selected from the group consisting oflecithin, bilayer forming lipids, cholesterol and mixtures thereof.
 4. Amethod according to claim 3, wherein the emulsifier is lecithin in anamount of 0.1 to 0.5%.
 5. A method according to claim 3, wherein thepolymerizing agent includes a calcium ion.
 6. A method according toclaim 5, wherein the polymer matrix precursor is a non-cytotoxic polymerselected from the group consisting of alginate, polysaccharides,polylysine, starch and mixtures thereof.
 7. A method according to claim6, wherein the polymer matrix precursor is an alginate.
 8. A methodaccording to claim 1, wherein the dispersion medium comprises ahydrophobic solvent or mixture of solvents having a Specific Gravity ofabout
 1. 9. A method according to claim 8, wherein the dispersion mediumis a 1:3 mixture of chloroform:hexane.
 10. A method according to claim9, wherein the polymerizing agent is CaCl₂, at a concentration of 0.01 Mto 1.0 M.
 11. A method according to claim 2, wherein the dispersionmedium comprises an oil and an organic solvent miscible with oil.
 12. Amethod according to claim 11, wherein the polymerizing agent is CaCl₂ ata concentration of 0.01 M to 1.0 M, and wherein the emulsifier islecithin at a concentration of 0.17 to 0.5%/v.
 13. A method according toclaim 12, wherein the oil is corn oil and the organic solvent isn-hexadecane, and wherein the polymer matrix precursor is an alginate.14. A method according to claim 13, wherein capsule volume, diameter andlength are controlled by adjusting the proportion of n-hexadecane, thusvarying the viscosity and specific gravity of the dispersion medium,with the ratio of corn oil to n-hexadecane being in a range of about 2:3to about 7:3.
 15. A method according to claim 13, wherein capsulevolume, diameter and length are controlled by adjusting the proportionof alginate, thus varying the viscosity of the suspended alginate phase,the alginate being in the form of a 1% aqueous solution.
 16. A methodaccording to claim 14, wherein the ratio is 3:2.
 17. A method accordingto claim 1, wherein the microparticle is a viable cellular biomaterialand wherein the polymer-encapsulated biomaterial so formed retains itsviability.
 18. A method according to claim 17, wherein the cellularbiomaterial comprises a fungal propagule.
 19. A method according toclaim 18, wherein the fungal propagules are conidia, spores or hyphalcells.
 20. A polymer encapsulated microparticle, comprising amicroparticle or microparticles encapsulated in a polymer matrixcapsule, the polymer forming the polymer matrix being biodegradable,non-cytotoxic and water dispersible, the capsule being of a teardropshape 10-400 μm in diameter and having a length of 40-200% longer thanits diameter.
 21. A polymer encapsulated microparticle according toclaim 20, wherein the polymer is an alginate.
 22. A polymer encapsulatedmicroparticle according to claim 21, wherein the microparticle is aviable cellular biomaterial, whose viability is maintained afterencapsulation.
 23. A polymer encapsulated microparticle according toclaim 22, wherein the biomaterial comprises a fungal propagule selectedfrom the group consisting of conidia, spores and hyphal cells.
 24. Apolymer encapsulated microparticle according to claim 20, wherein thepolymer matrtix is formed of a polymer selected from the groupconsisting of alginate, polysaccharide, polylysine, starch and mixturesthereof.
 25. A herbicidal composition comprising the encapsulatedmicroparticle of claim 20, wherein the microparticle is a viablepropagule of a weed pathogenic fungus.